CN101240023A - Preparation of heavy metal cadmium polyclonal antibody and enzyme-linked immunosorbent assay method - Google Patents

Abstract

The invention discloses the preparation of a heavy metal cadmium polyclonal antibody and an enzyme-linked immunosorbent assay method. The preparation method of the heavy metal cadmium polyclonal antibody comprises (A) synthesis of a complete antigen, which is the key to the preparation of the polyclonal antibody; (B) preparation of the antibody. The steps of the enzyme-linked immunosorbent assay method include: (1) Determine the optimal reaction concentration of the original coating Cd-ITCBE-OVA and the antibody by the square matrix titration method, and coat the 96 wells with the optimal concentration of the original coating Cd-ITCBE-OVA ELISA plate; (2) Indirect competitive enzyme-linked immunosorbent assay test; (3) Investigate the influence of various conditions on CI-ELISA, optimize the reaction conditions; (4) Establish CI-ELISA under the optimized reaction conditions, Draw a standard inhibition curve. The detection limit of the invention is 0.04 μg/L, the linear range is ^{10-1-10 3} μg/L, has specificity to cadmium, and is suitable for the determination of trace cadmium in environmental water samples. At the same time, it provides technical support for on-site rapid detection of heavy metal pollution accidents, so as to facilitate timely remediation and recovery of pollution accidents.

Description

Preparation of heavy metal cadmium polyclonal antibody and enzyme-linked immunosorbent assay method

technical field

The invention relates to the preparation of heavy metal cadmium polyclonal antibody and the antibody-based enzyme-linked immunosorbent assay method (ELISA).

Background technique

The pollution caused by heavy metals is called heavy metal pollution. At present, the most serious heavy metals to the environment are lead, cadmium, mercury, chromium and arsenic, referred to as "five poisons". After heavy metals enter the human body, they are difficult to excrete and accumulate gradually. When they exceed the physiological load of the human body, they will cause changes in physiological functions, lead to acute and chronic diseases, or cause long-term harm. In recent years, with the rapid development of my country's economy, more and more environmental accidents have occurred frequently. The main type of pollutants is heavy metal pollutants, especially food poisoning, drinking water poisoning, and sudden pollution accidents of water bodies. Therefore, it is of great significance to establish a fast and convenient heavy metal detection method for long-term monitoring of heavy metal pollution to control pollution, protect the environment and human life safety and health.

At present, the development direction of heavy metal detection in the world is sensitive, accurate, real-time, fast, good selectivity and wide application range. However, most of the traditional heavy metal detection techniques require large-scale or specialized instruments (atomic absorption spectrometry, atomic emission spectrometry, etc.) The need for rapid and online detection. Immunological detection methods use the specific binding of antigens and antibodies to selectively identify and measure analytes that can be used as antibodies or antigens. It is an important branch of the long-term development of immunology, and has the advantages of fast detection speed, low cost, simple and portable instrument, high sensitivity and strong selectivity. It has been widely used in clinical medicine, life science and the detection of organic and toxic substances in the environment. At the same time, it also provides a new idea for the detection of heavy metals, and effectively supplements the traditional heavy metal detection methods.

Since Reardan et al. published an article in Nature in 1985 and prepared monoclonal antibodies through metal-chelating agents for the first time, there have been more and more foreign studies on heavy metal immunological detection methods. So far, Cd ²⁺ , Co ²⁺ , U ⁶⁺ , Pb ²⁺ , Gu ²⁺ , Hg ²⁺ , Zn ²⁺ , and In ³⁺ metal-chelator complex antibodies were developed, and corresponding ELISA methods were established. Patent CN1769893 discloses a method for preparing the complete antigen of heavy metal Pb ²⁺ . Zhu Xiaoxia et al. prepared monoclonal antibodies to cadmium and lead, and established a corresponding enzyme-linked immunosorbent detection method. The above methods are all based on heavy metal monoclonal antibody research. At present, most of the researches on heavy metal immunoassays focus on monoclonal antibodies and corresponding enzyme-linked immunoassay methods, and there are relatively few studies on polyclonal antibodies. Compared with polyclonal antibodies, the preparation of monoclonal antibodies is more costly, longer and riskier. Although more clonal antibodies are slightly higher in specificity, as far as the established detection method is concerned, polyclonal antibodies can also meet the detection requirements, and the detection limit and sensitivity of the method are close to those of monoclonal antibody-based detection methods.

Literature search shows that only David K.Johnson abroad prepared polyclonal antibody to cadmium in 1999 with tetraethylenediaminepentaacetic acid anhydride as a chelating agent, and used fluorescence polarization immunoassay to detect cadmium (Johnson, D.K., A fluorescence polarization immunoassay for cadmium (II). Analytica Chimica Acta, 1999.399: p.161-172).

Contents of the invention

1. The technical problem to be solved by the invention:

Aiming at the problems existing in the existing heavy metal cadmium immunological detection method, the present invention provides the preparation of heavy metal cadmium polyclonal antibody and enzyme-linked immunosorbent assay method, prepares a kind of heavy metal cadmium polyclonal antibody, and through antibody antigen reaction, establishes An enzyme-linked immunosorbent assay method for the detection of heavy metal cadmium for the rapid detection of environmental water samples.

2. Technical solution:

Principle of the present invention: prepare polyclonal antibody of heavy metal cadmium, and establish an indirect competitive enzyme-linked immunosorbent assay method (CI-ELISA). The key to the immunological detection method of heavy metals lies in the preparation of anti-heavy metal antibodies, including polyclonal antibodies and monoclonal antibodies, and the key to the preparation of antibodies lies in the preparation of heavy metal immunogens. The basic principle of the heavy metal cadmium polyclonal antibody preparation and indirect competitive ELISA method is: one end of the selective bifunctional chelating agent is chelated with the heavy metal to form a heavy metal-chelating agent complex as a hapten, and the hapten is then chelated The other end of the mixture is coupled with a carrier protein such as bovine serum albumin (BSA), ovalbumin (OVA) etc. to form a heavy metal-chelating agent-protein complete antigen (immunogen and coating original). New Zealand white rabbits were immunized with immunogen to obtain polyclonal antibodies. The basis of CI-ELISA is the immobilization of antigens and enzyme labeling of antibodies. The antigen combined on the surface of the solid phase carrier still maintains its immunological activity, and the enzyme-labeled antibody retains both its immunological activity and enzyme activity. During the determination, the soluble antigen is coated on the microtiter plate, and the standard solution of a series of concentrations of the test substance is added to the mixed solution after mixing and incubating with equal volumes of the antibody. The test antigen in the mixed solution competes with the antigen on the surface of the solid phase carrier for the surface of the antibody. the binding site. Then add the enzyme-labeled antibody, which is also bound to the solid phase carrier through the reaction. At this time, the amount of enzyme on the solid phase is in a certain ratio to the amount of the standard solution of the analyte in the mixed solution. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the standard solution of the analyte in the mixed solution, so qualitative or quantitative analysis can be performed according to the depth of the color. The reaction equation for antigen preparation is:

Concrete technical scheme of the present invention is as follows:

The preparation method of heavy metal cadmium polyclonal antibody, its step comprises

(A) The synthesis of complete antigen, the synthesis of complete antigen is the key to the preparation of polyclonal antibody, with 1-(4-isothiocyanatobenzyl)-ethylenediaminetetraacetic acid (ITCBE) as a bifunctional chelating agent, using Isothiocyanate method and heavy metal coupling with carrier protein to obtain complete antigen, including immunogen Cd-ITCBE-BSA and coating agent Cd-ITCBE-OVA;

(B) Preparation of antibody, with immunogen Cd-ITCBE-BSA, select three purebred male New Zealand white rabbits of 1.5-2.0 kg as experimental animals, collect negative serum one week before the test, and routinely immunize the white rabbits, Blood was collected from the heart about 3 months after immunization, and serum was prepared, and the antibody titer was determined by indirect non-competitive ELISA method, and its specificity to metal cadmium was identified by indirect competitive ELISA method, and the final concentration of 0.01% thimerosal was added to the serum. Freeze at -20°C to obtain a polyclonal antibody to the heavy metal cadmium.

In step (A), the full-wave scanning of the complete antigen is carried out by using the ultraviolet absorption method to determine whether the antigen is successfully synthesized. The content of protein and heavy metal cadmium in the complete antigen were determined by Coomassie blue method and flame atomic absorption spectrophotometry, respectively.

Enzyme-linked immunosorbent assay method, the steps comprising:

(1) Determine the optimal reaction concentration between the original coating Cd-ITCBE-OVA and the antibody by square matrix titration, and coat the 96-well microtiter plate with the optimal concentration of the original coating Cd-ITCBE-OVA;

(2) Prepare cadmium standard solution with PBS solution containing EDTA, mix with the antibody of equal volume 1/2 optimum working concentration, add in the microtiter plate as reaction solution and carry out indirect competitive enzyme-linked immunosorbent assay test;

(3) Investigate the influence of chelating agent EDTA and its concentration, salt ionic strength, pH value, blocking solution and premixing time on CI-ELISA, and optimize the reaction conditions;

(4) Under the optimized reaction conditions, establish CI-ELISA and draw the standard inhibition curve.

The detection limit, detection range, precision and accuracy of the method, and other metals ^such as Pb ²⁺ , Ni ²⁺ , Mg ²⁺ , Ca 2+ , Cu ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , Hg ²⁺ and Fe ³⁺ cross-reaction rates were analyzed; tap water and Yangtze River water were spiked and recovered using the established method.

3. Beneficial effects:

The invention provides the preparation of heavy metal cadmium polyclonal antibody and an enzyme-linked immunosorbent assay method. On the basis of preparing the heavy metal cadmium polyclonal antibody, an indirect competitive enzyme-linked immunosorbent assay method for measuring cadmium content in environmental water samples is established. The detection limit of the present invention is 0.04 μg/L, the linear range is 10 ^-1 ~ 10 ³ μg/L, it is specific to cadmium, and it is compatible with Pb ²⁺ , Ni ²⁺ , Mg ²⁺ , Ca ²⁺ , Cu ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , and Fe ³⁺ have a cross-reaction rate of less than 1%, and a cross-reaction rate with Hg ²⁺ of 7.4%; %between. This method is suitable for the determination of trace cadmium in environmental water samples. At the same time, it provides technical support for on-site rapid detection of heavy metal pollution accidents, facilitates timely remediation and recovery of pollution accidents, and can be developed into an important detection method for other samples in the environment and agricultural food safety detection, which has great practical significance.

Detailed ways

The present invention is further illustrated by examples below.

Example 1

Antibody preparation

Step 1: Synthesis of complete antigen

Weigh 10 mg ITCBE and dissolve in 10 mL pH 9.5 sodium phosphate buffer solution to form a 2.275 mM metal chelator solution. 40 mg of BSA protein was weighed and dissolved in pH 7.4 PBS (137 mM NaCl, 3 mM KCl, 8 mM Na ₂ HPO ₄ , 1 mM KH ₂ PO ₄ ) buffer solution to form a 40 mg/mL protein solution. Cadmium (99.999%) was dissolved in hot nitric acid and diluted to 4.7mM for use.

Take 2.5mL ITCBE and 1.5mL 4.7mM cadmium solution to react to obtain Cd-ITCBE complex. The complex was added dropwise to 1 mL of 40 mg/mL BSA protein solution. The reaction solution was reacted at room temperature under slow stirring for 24 hours, then put into EDTA pretreated dialysis bags, dialyzed three times with PBS buffer, twice with pure water, and dialyzed at 4°C for three days to remove unreacted Cd, ITCBE and Cd-ITCBE complex. Accurately measure the dialysate, which is the immunogen Cd-ITCBE-BSA, and store it at low temperature. The original coated Cd-ITCBE-OVA was prepared in the same way.

The content of protein in the conjugate was detected by ultraviolet spectrophotometer. The content of cadmium was determined by atomic absorption method. Scan the dialysate with an ultraviolet spectrophotometer to analyze and identify the conjugate. The results indicated that the complete antigen was successfully synthesized.

The content of protein in the conjugate was detected by ultraviolet spectrophotometer. Take 0.5mL of antigen in a colorimetric tube and add 3.5mL of distilled water. The standard curve method was used for determination. sample. Atomic absorption spectrometry was used to measure the content of Cd ions in the antigen. The protein concentration in the immunogen Cd-ITCBE-BSA was 7.024mg/mL, and the cadmium concentration was 62.49ppm; the protein concentration in the coating original Cd-ITCBE-OVA was 5.27mg/mL, and the cadmium concentration was 8.866ppm.

Step 2: Antibody preparation and identification

Cd-ITCBE-BSA was used as the immunogen, and three purebred male New Zealand white rabbits of 1.5-2.0 kg were selected as experimental animals, and negative serum was collected one week before the experiment. The specific immunization procedure is as follows: For the first immunization, 1 mg of immunogen per mouse was mixed with an equal volume of Freund's complete adjuvant, emulsified into a water-in-oil structure, and then injected subcutaneously at multiple points on the back (around 20 o'clock). Three weeks later, they were boosted with the same dose of immunogen and an equal volume of incomplete Freund's adjuvant. After that, it will be strengthened once every two weeks, and blood will be collected from the ear vein to measure the titer one week after the strengthening. Dilute the immunogen with an equal volume of normal saline for the last time and inject directly into the marginal ear vein. Eight days later, blood was collected from the heart, and serum was prepared, thimerosal was added with a concentration of 0.01% by weight, and frozen at -20°C after aliquoting.

The antibody titer was measured by the indirect ELISA (I-ELISA) method to determine whether there is an antibody against the metal ion. The titers of the three white rabbits were compared, and finally the white rabbit serum with higher antibody titer was selected for the establishment of CI-ELISA detection method.

Indirect non-competitive ELISA (I-ELISA) method steps:

(1) Coating, 100 μL/well, overnight at 4°C, washed 3 times, and patted dry; (2) Blocked, 200 μL/well, incubated at 37°C for 1 hour, washed 3 times, and patted dry; (3) Added antiserum (respectively Dilute to 4000, 8000, 12000, 20000, 30000, 40000 times) 100 μL/well, incubate at 37°C for 2 hours, wash 3 times, and pat dry; (4) Add goat anti-rabbit enzyme-labeled secondary antibody (1:10000 dilution) at 37°C Incubate for 1 hour, wash 3 times, and pat dry; (5) add chromogenic solution for 15 minutes at 37°C. (6) 50 μL/well sulfuric acid solution with a concentration of 2M terminates the color reaction; (7) Measure the OD450 value with a microplate reader, set it to zero with a blank control, use negative serum as a control, and take the OD450 value of the well to be tested to be greater than or equal to the negative well 2.1 times as positive.

After determination, the titer of the antibody reached 1:128×10 ⁴ , which can be used for enzyme-linked immunosorbent assay experiments.

Example 2:

ELISA method

Step 1: Determination of Potency

Use the square matrix titration method to determine the optimal working concentration of the antigen and antibody: the coated antigen (Cd-ITCBE-OVA) is serially diluted to 1500 times, 2500 times, 5000 times, 10000 times the AB, CD, EF of the coated enzyme plate, GH line; use 1% OVA as blocking solution; antiserum was diluted to 20000 times, 40000 times, 80000 times, 160000 times, 320000 times and 640000 times respectively, and added to 1 2, 3 4, 5 6, 7 8 of the microtiter plate, 9 10, 11 12 columns; add goat anti-rabbit-horseradish peroxidase enzyme-labeled secondary antibody (1:10000, diluted with 1% OVA); develop color with TMB, stop with 2M sulfuric acid, measure OD450 value. The antigen-antibody concentration with an OD450 value of 0.8-1.2 was used as the optimal working concentration. Finally, the optimum coating original Cd-ITCBE-OVA and antibody concentrations were determined to be 1.05 μg/mL and 1:30×10 ⁴ , respectively.

Step 2: Establishment of ELISA method

Under the condition of the optimal working concentration of coating agent and antibody, an indirect competition ELISA method (CI-ELISA) was used to establish the detection method of antibody to heavy metal cadmium. Prepare cadmium standard solutions with concentrations of 0.00, 0.10, 1.00, 10.00, 100.00, and 1000.00 μg/mL in PBS solution of 1.0 mM EDTA, and mix with the antibody overnight for pre-inhibition. Draw the standard inhibition curve, and calculate the sensitivity (IC50) and the lowest detection limit (IC20).

The steps of the indirect competitive ELISA method are as follows:

(1) Antibody and standard sample (sample to be tested) premixed: dilute the antibody to twice the working concentration with PBS (for example, the working concentration is 1:10000, it should be diluted to 1:5000) and then mixed with an equal volume of standard sample (sample to be tested) Premix at room temperature.

(2) Coating: Dilute the coating material to the working concentration with CBS coating buffer, add 100 μL/well to the eight rows of AB, CD, EF, and GH of the 96-well ELISA plate, and overnight at 4°C.

(3) Blocking: add 200 μL of blocking solution to each well, and incubate at 37° C. for 1 h.

(4) Adding samples: Add the premixed liquid to the microtiter plate, 100 μL/well, and incubate at 37° C. for 2 hours.

(5) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody to the working concentration with blocking solution, add 100 μL/well to the enzyme-labeled plate, and incubate at 37°C for 1 hour.

(After each step above, wash 3 times with washing liquid)

(6) Color development: Add 100 μL/well of the substrate solution to the microtiter plate, and develop color for 15 minutes.

(7) Stop the reaction: 50 μL/well 2M H2SO4 is quickly added to the microplate, and the absorbance value is read at 450 nm.

Step 3: Condition optimization

Change the EDTA concentration in the metal standard solution dilution buffer so that the EDTA concentration in the reaction system is 0.1mM, 0.5mM, 1.0mM, 5.0Mm, 20mM, 20.0mM, 50.0mM, and compare the inhibitory effects of different EDTA concentrations on positive serum.

In the competitive ELISA reaction, under the condition of the optimal working concentration of antigen and antibody, change the salt ion strength in the matrix so that the concentration of NaCl in the reaction system is 0M, 0.05M, 0.15M, 0.5M, 1.0M, and compare the effect of different ion strength on Effect of ELISA detection curve.

The function of blocking solution is to eliminate non-specific adsorption. In the competition ELISA reaction, under the conditions of the optimal working concentration of antigen and antibody and the optimal salt ion strength, the present invention respectively adopted 1% gelatin, 1% OVA, and 3% skimmed milk powder as the blocking solution (200 μ L/hole). Its influence on the detection curve.

In the competitive ELISA reaction, under the conditions of the optimal working concentration of antigen and antibody, the optimal salt ion strength and the optimal blocker blocking conditions, the influence on the results of the competitive ELISA is reflected by changing the pH value in the reaction matrix. The pH values in the reaction system were 5.7, 6.5, 7.4, and 8.5, respectively, and the effects of different pH values on the ELISA detection curve were compared.

In the indirect competitive ELISA reaction, the premixing time of the metal standard solution and the positive serum was set at room temperature overnight and at room temperature for 1 hour, and the effects on the inhibition curve were compared.

Step 4: Method Evaluation

By determining the optimal conditions of the indirect competition ELISA method, the present invention uses 1:5000 (protein concentration of 1.05 μg/mL) coating original to coat the 96-well ELISA plate, and uses 1% gelatin as the blocking solution. The antibody concentration was 1:300,000. The concentration of chelating agent EDTA in the antigen-antibody reaction system is selected as 1.0mM, the pH value is 7.4, and the ionic strength is 0.15M NaCl. The premixing time of antibody and standard solution is 1 hour. as the best reaction conditions. Under the optimal working conditions, the inhibition curve was obtained, the inhibition concentration IC50 was 33.0ppb, the IC20 was 2.7ppb, and the ^R2 was 0.9976. Take Logit (B/B0) as the ordinate (Y), and the negative logarithm value of Cd ²⁺ concentration (ppb) as the abscissa (X), and make a standard curve. When Cd ²⁺ is in the range of 0.1ppb-1000ppb, there is a significant linear relationship between Logit (B/B0) and the logarithmic value of Cd ²⁺ concentration (ppb), and the regression equation Y=0.6834X+0.4107 is obtained, and the correlation coefficient reaches 0.998.

Under the above optimal reaction conditions, method-specific experiments were carried out. Prepare standard solutions of other metals such as Pb ²⁺ , Ni ²⁺ , Mg ²⁺ , Ca ²⁺ , Cu ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , Hg ²⁺ and Fe ³⁺ , the experimental operation is the same as step two. The cross-reaction rate with Pb ²⁺ , Ni ²⁺ , Mg ²⁺ , Ca ²⁺ , Cu ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Cr ³⁺ , Fe ³⁺ is less than 1%. The Hg ²⁺ cross-reactivity rate was 7.4%. The antibody is very specific to metal cadmium.

Take tap water and Yangtze River water to carry out standard addition recovery rate experiment, carry out the test under the optimal conditions determined in step 3, the operation process is the same as step 2. The spiked recoveries of tap water and Yangtze River water were 103.5%-116.3% and 85.5%-112.5%, respectively.

Claims (5)

1. the preparation method of a heavy metal cadmium polyclonal antibody, it may further comprise the steps:

(A) antigenic synthetic, with 1-(4-isothiocyano phenmethyl)-ethylenediamine tetraacetic acid (EDTA) as bifunctional chelating agent, adopt different sulphur hydrocyanic ester method and heavy metal cadmium and carrier protein couplet, obtain complete antigen, comprise immunogen Cd-ITCBE-BSA and coating antigen Cd-ITCBE-OVA;

(B) preparation of antibody and evaluation, with immunogen Cd-ITCBE-BSA, the purebred male new zealand white rabbit of choosing three 1.5-2.0kg is as experimental animal, test and gather negative serum the last week, the routine immunization method is carried out immunity to White Rabbit, immunity is the heart blood sampling after 3 months, preparation serum, measure antibody titer with indirect non-competing ELISA method, the indirect competitive ELISA method is identified its specificity to cadmium metal, it is 0.01% Thiomersalate that serum adds concentration, after the packing in-20 ℃ frozen, promptly obtain the polyclonal antibody of heavy metal cadmium.

2. the preparation method of heavy metal cadmium polyclonal antibody according to claim 1 is characterized in that described step (A) is antigenic to adopt ultraviolet absorption method that complete antigen is carried out all-wave scanning in synthetic, to determine whether synthetic merit of antigen.

3. the preparation method of heavy metal cadmium polyclonal antibody according to claim 1 is characterized in that described step (A) is antigenic to adopt beautiful blue laws of coomassie and atomic absorption spectrophotometry that the content of protein content in the complete antigen and heavy metal cadmium is measured respectively in synthetic.

4. enzyme-linked immunosorbent assay for measuring, its step comprises:

(1) adopts the square formation volumetry to determine the optimum response concentration of coating antigen Cd-ITCBE-OVA and antibody, wrap by 96 hole enzyme plates with the coating antigen Cd-ITCBE-OVA of optimum concn;

(2) with the PBS solution preparation cadmium standardized solution that contains EDTA, mix, add as reaction solution and carry out the test of indirect competitive enzyme-linked immunosorbent determining adsorption in the enzyme plate with the antibody of isopyknic 1/2 best effort concentration;

(3) investigate sequestrant EDTA and concentration, salt ion intensity, pH value, confining liquid, do time to the influence of CI-ELISA in advance, optimize reaction conditions;

(4) under optimized reaction conditions, set up CI-ELISA, drawing standard suppresses curve.

5. enzyme-linked immunosorbent assay for measuring according to claim 4, it is characterized in that sequestrant EDTA concentration is 1.0mM in the antigen antibody reaction system, ionic strength is 0.15M NaCl, matrix and liquid pH value to be measured are 7.4, with 1% gelatin is that doing time in advance of confining liquid, antibody and mark liquid is to carry out under 1 hour the optimum reaction condition, obtains competition and suppresses typical curve.